Alteration Of The Key Genes For Avermectin Biosynthesis In Streptomyces Avermitilis

Avermectins are 16-membered macrocyclic polyketides produced by Streptomyces avermitilis. They are known to be excellent anthelmintic agents with a good potency and broad spectrum of activity against a variety of nematode and anthropod parasites, and with a low level of side-effects on the host. Eight compounds are produced by S. avermitilis. Among these compounds, the B1a compound exhibits the most potent activity. Therefore, the research of eliminating undesirable metabolic products and enhancing the yield of useful component B1a is becoming a hotspot among many countries.In this study, aveC and aveD which were considered to be the key genes of avermectins biosynthesis, were altered by the method of gene engineering for the purpose of exploring.aveC encodes a dehydratase, and the high level of its expression could potentially enhance the ratio of "1" component to other components. In this paper, another aveC copy was inserted into the genomic DNA of S. avermitilis by gene manipulation, expecting to enhance the activity of dehydratase. By PCR amplification of the genomic DNA of recombinant strains, it was testified that the second aveC copy was inserted exactly into the expected site. However, the HPLC analysis of fermentation products produced by recombinant strains showed that the content of "1" component changed indistinctively. It implied that the activity of dehydratase may not be the limiting factor for avermectin biosythesis in S. avermitilis.aveD encodes a C₅-O-methyltransferase, which catalyze the coversion of "B" component to an useless "A" component. To eliminate "A" component, a disruption plasmid pCG23 was constructed by inserting an apramycin resistance gene into aveD gene fragment. Then the plasmid was introduced into S. avermitilis AVD2-14 by conjugal transfer. The double cross-over recombinant strain AV-C337 was selected and the fermentation products of this strain was analyzed. Results showed that it only produced two components of avermectins which were B_2a and B_1a respectively, and the useless "A" components completely disappeared. However, the yield of fermentation products of the recombinant strain obviously decreased.In order to avoid the decreasing of fermentation products caused by inserting long gene fragment, a mutation deleting five conservative amino acids was introduced into aveD by...